Studies on homotypic and heterotypic communications in chaperone protein ClpB from T. thermophilus

Abstract

ClpBTth is a molecular motor, which exerts an ATP hydrolysis driven mechanical force, resulting in disaggregation of aggregated proteins. ClpBTth belonging to the AAA family of ATPases carries the signature AAA module, which comprises of a large nucleotide binding domain (NBD) and an α-helical small domain (SD). The two tandem AAA modules present per subunit of ClpBTth interact with each other and the neighboring AAA modules in the hexameric ring-like structure. The current study focuses on inter-subunit (homotypic) and intra-subunit (heterotypic) communications between the AAA modules in ClpBTth oligomer, in respect to nucleotide binding and hydrolysis. The two tandem AAA modules of ClpBTth upon isolation exhibit unique properties. The isolated AAA2 module more or less represents a building block for the full-length hexameric protein. It appears to have retained most of the key characteristics, exhibited by full-length ClpBTth, as evident by sigmoidal kinetics in ATP hydrolysis and nucleotide binding-related conformational changes. So, nucleotide binding in the isolated AAA modules was investigated using fluorescently labeled proteins to gain insights into the nucleotide-mediated oligomer dissociation. Experiments were performed using the isolated AAA modules to reduce the complexity which comes with studying full-length hexamer. Experiments provided hints for involvement of the α-helical small domain 2 in nucleotide-dependent oligomer formation. Importance of the presence of SD2 has been demonstrated; upon its deletion, isolated AAA2 domain lost nucleotide binding and hydrolysis. Studies using a mutant carrying proline mutation in a loop connecting SD2 to NBD2 in the AAA2 module revealed loss of chaperone and ATPase activity. This study pointed out at the importance of flexibility and motion in SD2 of the AAA2 module. Nucleotide binding studies hinted at a possible biphasic nature and inter-subunit communication. ADP binding in one AAA2 module appeared to have triggered a conformational change in SD2 of the neighboring AAA2 module. These results gave insights into the conformational changes involved in ADP-mediated oligomer dissociation in ClpBTth. Although oligomer dissociation has been linked to ADP binding/formation in several instances, the inter-subunit communications pattern has never been clearly discerned. This work provides a platform for further studies to investigate conformational changes that result in oligomer formation and dissociation. The isolated AAA modules of ClpBTth upon reconstitution exhibit functional higher order oligomer formation. This reconstituted complex resembles wild type ClpBTth hexamer in all aspects related to oligomerization, chaperone activity and ATP hydrolysis. So, complex formation between the isolated AAA modules was studied to understand the thermodynamics behind the communication between them. Isothermal titration calorimetry measurements were performed to study binding between the isolated AAA modules. Experiments provided hints at temperature dependency in binding between the AAA modules in ClpBTth. Allostery in ATP hydrolysis is central to the function of ClpBTth, which represents both homotypic and heterotypic communications between the AAA modules. Absence of heterotypic allostery always resulted in a loss of function in ClpBTth. Importance of heterotypic allosteric communications between the AAA modules within each subunit and their role in chaperone activity of ClpBTth was investigated in this work. This was done by alteration of the related interface by mutating amino acids involved in interface interactions. Most of the mutants resulted in subtle changes in nucleotide hydrolysis properties and heterotypic allostery. Changes in allosteric behavior did not translate into a loss in chaperone activity, as evident by no loss of function in mutants exhibiting altered allostery. Experiments in the presence of GdmCl, which acts as an uncompetitive inhibitor for ATP binding, additionally revealed interesting insights to the allostery-defective situation in ClpBTth. Furthermore, this study has shed some light on possible mechanisms involved for attaining catalytic effectiveness in ClpBTth.